Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8 (HHV-8), is a human pathogen responsible for several AIDS associated malignancies including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Increasing evidence suggests that the lytic life cycle of the virus plays a critical role in the development of KS and other KSHV-associated diseases. In KS lesions, constant lytic replication sustains the population of latently infected cells that otherwise are quickly lost by segregation of latent viral episomes as spindle cells divide. Lytic DNA replication of herpesviruses requires both cis-acting element, the origin, and trans-acting factors including virally encoded origin-binding proteins, core replication machinery proteins and cellular factors. In addition, some epigenetic effects such as nuclear location, chromosome structure and RNA transcription influence DNA replication. To understand KSHV lytic DNA replication, we recently identified two ori-Lyts in the KSHV genome and characterized several critical cis-acting elements in the ori-Lyts. Furthermore, two regulatory proteins, K8 and RTA, were found to bind to the origins and their roles in initiation of DNA replication were investigated. These discoveries set a foundation for further investigation into the mechanism of KSHV lytic DNA replication that will be explored in the studies proposed here. In next funding period, emphasis will be placed on initiation of KSHV ori-Lyt-associated DNA replication. (1) We will characterize KSHV DNA replication complex. Roles of K8 and RTA in the complex formation or recruitment of the complex onto ori-Lyt will be investigated. (2) Many cellular proteins that might be involved in KSHV DNA replication have been identified. Two cellular proteins that may involve origin DNA unwinding and nuclear matrix attachment will be further studied because these two events are believed to be antecedent steps before replication fork formation. (3) We will study interplay between ori-Lyt-associated transcription and DNA replication. We will test the hypothesis that the transcription alters either DNA or chromatin structure and facilitates initiation of DNA replication. The results will greatly enhance our understanding on mechanisms that regulate initiation of KSHV replication. In addition, the studies will lead to new therapeutic strategies to block KSHV DNA replication and to treat KSHV-related diseases. This proposal represents the effects of our laboratory in understanding mechanisms that control and regulate KSHV lytic DNA replication. We will investigate the initiation of DNA replication in three aspects, i.e. formation of viral replisomes and their recruitment onto ori-Lyt, involvement of cellular factors in viral DNA replication and regulation of viral DNA replication by epigenetic effects. The study aims to new therapeutic strategies to block KSHV replication and treat KSHV-associated human diseases PUBLIC HEALTH RELEVANCE This proposal represents the effects of our laboratory in understanding mechanisms that control and regulate KSHV lytic DNA replication. We will investigate the initiation of DNA replication in three aspects, i.e. formation of viral replisomes and their recruitment onto ori-Lyt, involvement of cellular factors in viral DNA replication and regulation of viral DNA replication by epigenetic effects. The study aims to new therapeutic strategies to block KSHV replication and treat KSHV-associated human diseases.